Guidelines for the description of rhizobial symbiovars

Abstract Rhizobia are bacteria that form nitrogen-fixing nodules in legume plants. The sets of genes responsible for both nodulation and nitrogen fixation are carried in plasmids or genomic islands that are often mobile. Different strains within a species sometimes have different host specificities, while very similar symbiosis genes may be found in strains of different species. These specificity variants are known as symbiovars, and many of them have been given names, but there are no established guidelines for defining or naming them. Here, we discuss the requirements for guidelines to describe symbiovars, propose a set of guidelines, provide a list of all symbiovars for which descriptions have been published so far, and offer a mechanism to maintain a list in the future.


IntRoductIon to symbIoVaRs
The outstanding characteristic of rhizobia is their capacity to form nitrogen-fixing nodules in legume plants.The genes involved in this process have been studied for a long time, especially the nod genes involved in nodulation [1].These nod genes, as well as nif and fix genes (involved in nitrogen fixation), are found in symbiosis plasmids or islands, together with other symbiosis genes and genes that encode diverse secretion systems [2,3].Notably, symbiosis genes constitute a small fraction of all genes found in symbiosis plasmids or islands, which contain other genes expressed in free-living conditions [4] or for environmental adaptability [3,5].
Symbiosis genes are part of the accessory and mobile bacterial genome that may be gained by horizontal gene transfer (HGT) or lost [6][7][8].Indeed, there are many non-symbiotic natural isolates in the same species as rhizobia [9][10][11][12][13].The transfer of symbiosis plasmids in the laboratory can make non-symbiotic isolates symbiotic [14][15][16][17][18] and may convert pathogenic bacteria into symbionts [19].Root exudates, including plant flavonoids, may enhance the transfer of plasmids or islands between rhizobia [20,21] and transfer may be conditioned by bacterial density sensed by quorum sensing systems [22].In phylogenetic trees, incongruent phylogenies of symbiosis and core genes are indicative of the natural lateral transfer of symbiosis genes among rhizobia.Consequently, the species name is not indicative of the symbiosis capabilities, although many early names were based on this assumption.The mobility of symbiosis genes means that strains of different species may have the same symbiosis genes and phenotype, while other strains of the same species may have different ones.Discussion and understanding of this situation have been facilitated by giving names to these distinct symbiosis variants, or 'symbiovars' .
The symbiovar concept can be traced to Jordan's description of the genus Rhizobium in the 1984 edition of Bergey's Manual [23].Considering the species Rhizobium leguminosarum, which nodulated legumes in the tribe Vicieae, Rhizobium trifolii, which nodulated members of the genus Trifolium, and Rhizobium phaseoli, which nodulated members of the genus Phaseolus, Jordan noted that they had no consistent distinguishing characteristics other than host specificity, and there was good evidence that host specificity was conferred by plasmid-borne genes that were transmissible between strains [14][15][16].He treated them as a single species, R. leguminosarum, with three host-specific biovars.Biovars proved useful for describing host-specificity variants in other rhizobial species, and many were described over the subsequent decades.Eventually, Rogel and collaborators proposed that 'biovar' should be replaced by the more specific term 'symbiovar' , indicating that it was based on symbiosis specificity for the plant [24].A symbiovar is characterised by a distinctive set of genes that confer on rhizobia the capability to form nodules in particular plant(s).Single nod or nif genes would not alone be responsible for the specificity phenotype, but are used as markers representing the set of genes responsible [25].
In pathogenic members of the genus Agrobacterium, the term 'biovar' was used to refer to genetically distinct species, some of which have been reclassified, such as biovar 2 which is now Rhizobium rhizogenes [26,27].As rhizobia are close relatives of Agrobacterium, it was useful to clearly define the term symbiovar (symbiotic variant) in rhizobia as distinct from biovar (biological variant) [24].
The term pathovar is used for pathogenic bacteria to indicate the host affected by a particular species.For pathogenic bacteria, international standards for naming pathovars have been published [28], but there have never been any similar standards for symbiovars.It would be helpful to the rhizobial research community to establish some criteria for the description of symbiovars and to maintain a list of published names.The International Committee for Systematics of Prokaryotes (ICSP) Subcommittee for Rhizobia and Agrobacteria, of which we are members, has tasked us with defining suitable criteria and has agreed to host the list [29].

consIdERatIons foR a stablE classIfIcatIon of symbIoVaRs the components of a classification system
The requirements for a stable nomenclature of symbiovars should be essentially similar to those for naming bacterial species.It is important to be clear, though, that symbiovars are not part of bacterial taxonomy and are not covered by the International Code of Nomenclature of Prokaryotes [30].Nevertheless, we still need guidelines for defining symbiovars, naming them and assigning priority.We need to check that the guidelines have been followed, and provide an up-to-date list of accepted symbiovars, with references.Our proposed guidelines are given later in this article, after a discussion of some relevant considerations.

Recognising symbiovars
Differences in the specificity of interaction with plant hosts were the original rationale for symbiovars, and information on nodulation remains key for their definition.Information on nitrogen fixation and/or other effects on plants is also valuable.Many of the rhizobial genes involved in host specificity have been studied [1,2,31], but others remain to be defined and could be identified from genome sequences or the results of experimental analyses.It makes sense to include an analysis of symbiosis genes as part of the description of the symbiovar.This is particularly important because some host plants can be nodulated by rhizobia carrying very different symbiosis genes, so a shared host range alone does not guarantee that the genetic basis is similar.If bacteria of different species nodulate the same host, it is important to establish whether they have closely related symbiosis genes that have been passed around by horizontal gene transfer (in which case, it is justified to consider them to be the same symbiovar), or have independently converged on the same host specificity using different genes (in which case it is better to describe them as distinct symbiovars).Distinctive sets of symbiosis genes, and their phylogeny, may also be important for distinguishing symbiovars in the case of rhizobia that have very broad host ranges, as it can be difficult to define differences in host specificity.Sequences and phylogenies of symbiosis genes (nod and nif) can be used to identify symbiovars [24].In bacterial taxonomy, average nucleotide identity (ANI) of the whole genome has proved to be very useful to distinguish distinct species and ANI has become a gold standard [32].Species boundaries are usually around 95-96 % ANI, but need to be assessed in each case by seeking a minimum in the distribution of ANI values [33].Similarly, the percentage identity in nucleotide or amino acid sequences of marker symbiosis genes can help to distinguish between distinct symbiovars, but it is not appropriate to set thresholds for individual genes because specificity is determined by the ensemble of nod (and, to a lesser extent, nif) genes, and not all genes need to be highly diverged.Nevertheless, it is generally true that the symbiosis genes evolve together, and each gene will show less variation within than between symbiovars.Hence, phylogenetic trees of representative genes allow distinct groups to be delineated.Several different individual genes have been used for this purpose, though the majority of authors have included nodC and we suggest that a phylogeny of this gene should be included in the description of every new symbiovar.In the genus Bradyrhizobium, a limit of 92.5 % identity of nodC gene sequences served to distinguish symbiovars sojae and pachyrhizi [34], and a 92 % threshold was suggested for symbiovars in the genus Paraburkholderia [35], similar to the value proposed for symbiovars of Ensifer fredii [36].For a number of other symbiovars in the genus Bradyrhizobium, a limit in NifH amino acid identity around 95 % was found to be suitable [25].However, these genes are not the unique determinants of specificity, as there are other genes involved.Besides the common nod and nif genes, rhizobial secretion systems, especially type 3 but also type 6, are known to have important roles in host specificity [2,31], so it would be advisable to include them or any other relevant symbiosis gene [37] in the symbiovar description.Since it is now almost as easy to sequence entire bacterial genomes as to sequence individual genes, we think that each symbiovar should be represented by at least one complete genome sequence, so that all the potential host specificity determinants can be explored.It is important that the genome sequence should be of good quality, just as for species descriptions [38], and authors should check that the symbiosis gene sequences, especially of nodC, are complete and reliable.For the symbiovars described to date, we have, where possible, selected a representative strain with a sequenced genome, and this should be a requirement for symbiovar description in future.
All symbiovars described so far have a representative strain for which at least the nodC sequence is published (and in most cases the genome sequence), with one exception.The representative strain defined by the authors of sv.medicaginis is CI4, but they only provided a sequence for nodA, not for nodC [39].There is another strain of sv.medicaginis, USDA1170, which appears in the original authors' nodA tree and has an identical nodA sequence to that of CI4.There is a genome sequence available for this strain (GCF_004003925.1),so the nodC sequence is available and could be used to represent sv.medicaginis in future studies.We have included the genome accession of USDA1170 in Table 1, but have not proposed that this strain should replace CI4 as the representative strain because its host specificity has not been determined experimentally.

naming symbiovars
Symbiovar names proposed so far are in a Latin style similar to species epithets.Symbiovar is not a taxonomic category at all, but a description of a phenotype that is conferred by a set of genes that is potentially mobile between species and even between genera.Thus, symbiovar names can be considered to describe the word 'symbiovar' .For example, 'symbiovar lancerottensis' is the symbiovar from Lanzarote, whichever rhizobial genus it occurs in.Since the word 'symbiovar' is derived from the Latin 'varietas' , which is feminine, we propose that all symbiovar names should reflect the feminine gender regardless of the bacterial genus they are found in.In the past, symbiovar names were proposed that agreed with the gender of the genus, which was most often neuter.Rather than make an immediate 'correction' of these names, we list them in Table 1 both in the familiar neuter form (e.g.'-ense') and in the feminine form (e.g.'-ensis') that we think is more appropriate for the future.We consider the two forms to be synonymous.
There are a few symbiovar names that, if they had been proposed as species epithets, would have been corrected by the nomenclature editors because they are not well formed according to the rules for Latin names.Thus, lysilomaefficiens [25] would be better as lysilomefficiens (based on the stem Lysilom-of the host plant name), phaseolarum [40] would be phaseolearum (of the tribe Phaseoleae), oxytropis [41] would be oxytropidis (of Oxytropis), maamori [42] would be maamorensis (from the place Maamora).While authors may, in future, prefer to use the corrected form, we consider either version to be acceptable.Symbiovar names should be unique, even those that are found in different genera, because one of their key features is that they can be shared between species and even between genera.We need to resolve several cases of duplicate names that are already published.The name orientalis/orientale (differing only in gender) has been used twice, first for strains of Neorhizobium galegae that are symbionts of Galega orientalis [43] and later for Rhizobium mongolense and Rhizobium yanglingense symbionts of various wild legume species [44].To resolve this, we propose to change the name of the later symbiovar to sv. polyphytorum, since it has been isolated from many different host plant species.The name sv. tropici has also been used twice, first for broad host-range strains of Rhizobium tropici, Rhizobium leucaenae and Rhizobium lusitanum [5] and later for Bradyrhizobium symbionts of Centrosema species and other legumes [40].We propose that this second symbiovar be renamed sv.tropiciagri, since Bradyrhizobium tropiciagri is one of the species in which it is found.The name sv. mediterranense was defined for Sinorhizobium symbionts of Phaseolus vulgaris [45,46], but later the same name was used to describe a strain of Phyllobacterium sophorae that nodulated Lupinus micranthus in Tunisia [47], and this last symbiovar was also found later in Microvirga tunisiensis strains from both Tunisia and Morocco that nodulate various Lupinus species [48,49].We propose that this later symbiovar be renamed magrebensis, reflecting its isolation from two countries in the Maghreb region (northwest Africa).The name sv. mimosae was first used to describe strains of R. etli that nodulate members of the genus Mimosa [50,51], but the same name was later applied to strains of Paraburkholderia that also nodulated Mimosa species but using very different symbiosis genes, so they are clearly a different symbiovar [52].We propose that the symbiovar found in Paraburkholderia be renamed sv.mimosoidearum (of the traditional subfamily Mimosoideae).
We also need to resolve cases where the same symbiovar has been defined with different names, as is the case of symbiovar hedysari/aridi, defined in Mesorhizobium camelthorni first as sv.hedysari [53] and some months later as sv.aridi [54].In this case, considering priority for the date of definition, we should maintain hedysari and consider aridi as a later synonym.Hence, sv.aridi is not listed in Table 1.
The proposal of a new symbiovar name needs to be accompanied by a discussion of the evidence that it is novel and distinct, including information on host specificity and on gene sequences.The names sv.albiziae, sv.robiniae, sv.sophorae and sv.astragalus/ caraganae have been used to label clades in a nodC phylogeny [55], but the authors did not describe them in the text, so we consider that they did not define these potential symbiovars and we have not included the names in Table 1.Several later publications have repeated the names [41,42,56], but still with no formal definition.
When Jordan [23] first described biovars in R. leguminosarum, he used italics for the names, but when Rogel et al. [24] introduced the more specific term symbiovar they did not use italics.Both styles have been followed extensively in the literature, and we consider that it is equally acceptable to write symbiovar names with italics or without.1b.A symbiovar must have a representative strain, and the genome sequence of that strain must be publicly available and of sufficient quality [38].It is recommended (but not obligatory) that the representative strain be available in an international culture collection.
1c.It must be demonstrated that the symbiosis genes are recognisably distinct from those of all previously described symbiovars in sequence or in the presence/absence of specific genes.naming 2a.The of a new symbiovar must be distinct from all previously recognised symbiovar names in all genera.
2b. Symbiovar names that are adjectival always take the feminine gender.
2c.In other respects, the names of symbiovars follow the same rules as those of species epithets.3c.New symbiovars that conform to the guidelines and have priority will be added to the online list of recognised symbiovars on the Subcommittee website (currently https://sites.google.com/view/taxonomyagrorhizo/home).
3d.The priority of names is established by the date of inclusion in the list.

lIst of REcognIsEd symbIoVaRs
A list of all symbiovars (in alphabetical order) proposed to date is given in Table 1.For each symbiovar, references are given for the name and description, for the rhizobial species in which it has been found naturally, and for the range of legume hosts that are nodulated.In most cases, the original authors did not specify a representative strain; whenever possible, we have selected one with an available genome sequence.

conclusIon
In view of the large number of symbiovars described so far, and the likelihood that the number will increase in the future when more legumes are thoroughly studied, we felt the need to consider some appropriate standards and to clarify the status of the symbiovars already described.We have proposed guidelines for the description and naming of symbiovars and offered to set up and maintain a public list as a service to the rhizobial research community.Our aim is to make it easier to understand and use the symbiovar concept.As with any new venture, we may find we need to make adjustments in the light of experience, and we welcome feedback from users.
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Table 1 .
The list of recognised symbiovars

guIdElInEs foR thE dEscRIPtIon and namIng of symbIoVaRs description 1a
. Plant specificity is the essential basis to define a symbiovar.Symbiovars should be described in relation to the symbiosis phenotypes, not only referring to nodulation but also to effectiveness by considering nitrogen fixation.

Table 1 .
Continued2d.A proposal for a new symbiovar must be published in the body (not supplementary files) of an article in a peer-reviewed journal, and must include all the information required by these guidelines.Recognition3a.The ICSP Subcommittee for Rhizobia and Agrobacteria undertakes to maintain a list of recognised symbiovars and to appoint persons to manage this.3b.After publication, authors should submit the publication to the Secretary of the ICSP Subcommittee for Rhizobia and Agrobacteria.